Abstract: The Bcr-Abl oncoprotein is present in nearly all chronic myelogenous leukemia (CML) patients and 15-30% of acute lymphoblastic leukemia (ALL) cases. Here we hypothesize that FOXO3a serves as a tumor suppressor in hematopoietic cells and that Bcr-Abl expression removes this suppressive function by mediating the phosphorylation and subsequent proteasomal degradation of FoxO3a, thereby promoting cellular transformation. Thus, FOXO3a represents a potential therapeutic target for the treatment of myeloproliferative disorders. We base this hypothesis on the observations that 1) FOXO3a functions to regulate Bcr-Abl-induced evasion of apoptosis; 2) Bcr-Abl mediates the inhibition of FOXO3a activity in a proteasome degradation- dependent manner; 3) a -TrCP1 E3 ligase and a PP2A phosphatase are putative regulators of FOXO3a stability; 4) inhibition of Bcr-Abl by kinase inhibitors frequently leads to resistance, due to mutations of Bcr-Abl; 5) therapeutics that target the downstream effector molecules can be effective inhibitors that will not result in selection pressure for resistant Bcr-Abl mutations; and 6) treatment with the highly specific proteasome inhibitor, bortezomib, restores levels of FOXO3a and consequently decreases the survival of Bcr-Abl- expressing cells and a Bcr-Abl-positive leukemia patient. Our Aims are: Aim 1: To determine whether Bcr-Abl-induced down-regulation of the FOXO3a transcription factor in hematopoietic cells is critical for cellular transformation. We will investigate the involvement of FOXO3a as a tumor suppressor in A) mice bearing hematopoietic cells expressing Bcr-Abl and a wild-type (wt) or a constitutively active FOXO3a; B) primary bone marrow cells from FoxO3-null or control mice retrovirally transduced with Bcr-Abl alone or in combination with wt or constitutively active FOXO3a; C) in FoxO3a null mice. Specific Aim 2: To investigate the molecular mechanism for Bcr-Abl-induced regulation of FOXO3a: targets for cancer therapeutics. We plan to investigate the role of -TrCP1 and PP2A in Bcr-Abl-induced regulation of FOXO3a by immunoprecipitation and immunoblot analysis, site-directed mutagenesis and shRNA techniques. We also plan to design and analyze peptide mimetics that can stabilize FOXO3a. Specific Aim 3. To determine whether upregulation of FOXO3a by the highly the highly specific proteasome inhibitor bortezomib is critical for its therapeutic effect. As a specific FOXO3a-based therapy is far from reality, current therapeutic strategies that target FOXO3a could have immediate translation to the clinic. Here, we will investigate the requirement of FOXO3a for mediating the therapeutic efficiency of bortezomib in Bcr-Abl-induced leukemia. Research Narrative: FOXO3a, a member of the Forkhead transcription factor family, regulates the expression of key apoptotic genes and is thought to be a tumor suppressor. We and others have established that Bcr-Abl, the primary cause of Chronic Myelogenous Leukemia (CML), inhibits FOXO3a to mediate evasion of apoptosis and to promote the survival of the transformed hematopoietic cells. In this proposal, we will test the hypothesis that FOXO3a is a tumor suppressor in hematopoietic cells and Bcr-Abl-expression via regulation of FOXO3a activity removes this tumor suppressor to promote cellular transformation. In particular, we propose that Bcr- Abl-induced regulation of the reversible phosphorylation and proteasomal degradation of FOXO3a will inhibit tumor-suppressor activity of FOXO3a in Bcr-Abl-expressing hematopoietic cells, potentially hematopoietic stem cells, and this leads to subsequent survival and leukemogenesis.